Immunodiagnostic test cards having indicating indicia

ABSTRACT

An immunodiagnostic test card includes a plurality of transparent chambers wherein each chamber includes a quantity of testing material that combines with a patient sample, when mixed, to produce an agglutination reaction. A plurality of indicia are disposed to aid in the manufacture and determining the usability of the cards prior to test and also in objectively grading the agglutination reactions that are formed or lack of agglutination.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.12/022,349, filed on Jan. 30, 2008 now U.S. Pat. No. 8,058,073, issuedon Nov. 15, 2011, the entire contents of which are herein beingincorporated by reference.

FIELD OF THE INVENTION

The invention relates to the field of immunodiagnostic testing and moreparticularly to an immunodiagnostic test card having a plurality oftransparent chambers used for testing a patient sample and producing anagglutination reaction, the card further including indicia forobjectively grading each reaction.

BACKGROUND OF THE INVENTION

So-called “gel” cards or “bead” cassettes are now commonly used, forexample, in the field of immunohematological testing as test elementsfor blood typing, blood grouping and/or the detection of certainantigens or antibodies. These test elements are commonly defined by aflat planar substrate having a plurality of transparent microtubes orcolumns that define test chambers. A predetermined quantity of inertbead or gel material is added to each of the microtubes. This inertmaterial may be coated with an antibody or an antigen or provided with acarrier-bound antibody or antigen or with specific reagents. Typically,a foil wrap is used to cover the top of the card or cassette, therebysealing the contents of each microtube until the time of test. The foilwrap is pierceable or otherwise removed to enable aliquots of patientsample and/or reagents to be added to each of the microtubes, eithermanually or in an automated apparatus. The sample is incubated and thenmixed into the contents of the test chambers by centrifugation. Duringcentrifugation, red blood cells (RBCs) in each reaction chamber arepulled into the gel column. Agglutinated RBCs are too large to passthrough the gel matrix, depending on the size of the agglutinates, whileunagglutinized RBCs will pass easily through the gel and pellet at thebottom of the chamber.

A grading system is used with regard to a resulting agglutinationreaction with regard to RBC agglutinates that are trapped anywhere inthe gel column. Positive reactions can be graded from 0 to 4+. Morespecifically, a 4+ reaction is indicated by a solid band of RBCs on topof the gel. A 3+ reaction displays agglutinated RBCs in the upper halfof the gel column. A 2+ column is characterized by RBC agglutinatesdispersed throughout the length of the column. A 1+ column is indicatedby RBC agglutinates mainly in the lower half of the gel column with someagglutinated RBCs being pelleted at the bottom of the column. Negative(0) reactions are characterized by a pellet of RBCs on the bottom of themicrotube with no agglutinates along the length of the column. In thecourse of testing, the resulting reaction can be highly positive; thatis, all or most of the formed agglutinate is disposed above the inertmaterial layer, or highly negative; that is, in which no agglutinationresults and all of the cells is located at the bottom of the microtubeas a pellet. Gradients of these reactions are also produced whereinformed agglutinate can be distributed anywhere throughout the gel/beadmatrix and wherein this distribution must be graded as being eitherhighly or weakly positive.

The grading of agglutination reactions using immunodiagnostic testcards, such as those manufactured by DiaMed, Inc. and Micro-TypingSystems, Inc., among others, is somewhat difficult, for agglutinationreactions, for example that are not highly positive or highly negative.Among the most difficult to grade are those reactions between a 4+ and a3+ reaction and between a 1+ and a negative (0) reaction. Ultimately,such determination becomes highly user-dependent especially when thetest cards or cassettes are read manually, making the process extremelysubjective as to the position of agglutinates in the column, andrequiring users who have significant experience perform the readingoperation in order to obtain consistent results. In addition and becausethe card or cassette surface is typically relatively smooth throughout,it is often difficult for image processing algorithms of automatedapparatus to accurately locate the exact position of the column(s) inthe field of view. Exemplary image processing algorithms are described,for example, in EP Patent 0 637 744 to Shen et al. This perceiveddifficulty drives additional cost into the design of a suitableillumination system. Having an incorrect determination can produce direor fatal consequences; for example in instances for determining properblood samples for transfusion.

The manufacturing of test elements as described above is also dependentupon adding the proper amount of inert material into each of themicrotubes. Though this manufacturing process can be automated, errorscan still result. For example, a dispensing mechanism can be improperlypositioned relative to the test element or tolerance buildup issues mayhave occurred in the manufacture of a card or cassette, requiringin-process manufacturing checks for fill volume and label placement. Ifan improper amount of inert material is provided in any of themicrotubes, the results as graded from the resulting agglutinationreaction may not be accurate. For those reasons, among others, it isdesirable to improve the design of immunodiagnostic test cards andcassettes.

SUMMARY OF THE INVENTION

Therefore and according to one aspect, there is described a method ofmanufacturing an immunodiagnostic test element, and more specifically atest card or cassette. The manufacturing method comprises the steps of:forming a planar substrate, said substrate including at least onesupported transparent well; providing a plurality of indicia on said atleast one transparent well; and adding a volume of testing material tothe interior of said at least one well. The indicia is used according tothis method to indicate a proper amount of material has been added toeach of the reaction wells. This indicia can be added by integrallymolding it into the transparent wells or the card/cassette itself or byother means in which the indicia can be located directly on the columnsor adjacent thereto. The indicia can be in the form of a plurality oflinearly spaced lines or other forms can be used to provide theuser/manufacturer with visual indicators.

According to another aspect, there is described a method for grading anagglutination reaction using an immunodiagnostic test card, said testcard including at least one column retaining a test material, the methodcomprising the steps of: providing a set of indicia relative to eachsaid at least one column of a said test card; adding patient sample andpossibly reagent to each column, mixing the patient sample with thecontents of the column to produce an agglutination reaction between thepatient sample and said test material, and grading the reaction byvisually noting the position of formed agglutinates within said columnrelative to said indicia. This indicia can be provided, according to oneembodiment, directly to the external surfaces of each column to providea measurement scale or visual reference for the user or for an automatedsystem. The indicia can take one of several forms; for example,indicator marks can be provided at predetermined locations along thecolumn, either directly within the column or adjacent thereto. Theindicator marks can be molded directly into the column or alternativelycan be separately applied, such as by printing, laser etching or othersuitable means to provide a more accurate gradation as to the positionof formed agglutinates within the column. The indicator marks can belinear in shape or can assume one of several convenient forms whereinthese marks can be clear or alternatively be colored in order to bettervisualize the position of agglutinate relative to a contained testmaterial matrix. In another example, the indicia can include one or moreraised or depressed marks, creating distinct relief from the smooth cardsurface that is more easily discerned by the illumination system of anautomated machine vision apparatus as a visual datum, for example, usinglines of known length and separation. This indicia assists the imageprocessing algorithms of an automated apparatus to locate or “find” thecolumns of the card in the visual field. The image processing algorithmscan then accurately determine the position of agglutinates relative tothe indicia and properly calculate a reaction grade. The indicia asraised marks also breaks off the smooth surface of the card and providesa transition point for light emitted from the illumination system of theapparatus to reflect. This reflection will be more easily observed as anedge by the image processing software of the apparatus, thereby reducingthe cost and complexity of the illumination system.

One advantage realized by the test card design described herein is thatsubjectivity and “skill” in the reading of test elements, andparticularly manual reading of same, is virtually eliminated. That is,manual reading of a test element is improved by providing a reliablevisual reference and measurement scale, thereby producing improved andrepeatable results and accuracy.

Another advantage is that test cards and cassettes can be manufacturedmore efficiently than previously known versions. The indicator marks orother indicia that is provided can be used with either in-process visualor machine vision systems to more easily determine the accuracy of thefill volume of gel or beads (or other suspended matrix) in the columns.The indicator marks or other indicia also provide a consistent datum onthe card or test cassette to assist in process visual systems todetermine if product labels have been correctly positioned on the testcard. In addition, incorporation of indicia as described herein can alsobe used by automated apparatus in order to reject cards in advance thatdo not include the indicia as a measure of quality assurance.

Yet another advantage is that the indicia used can also provide anindication relating to evaporation of contained test materials followingmanufacture of the test element that can readily deduced, thereby betterinsuring the quality of tests that are conducted.

These and other features and advantages will be readily apparent fromthe following Detailed Description, which should be read in conjunctionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a prior art immunodiagnostic test card following testingthereof, the card including several test chambers or columns, whereineach of the columns provide indication of an agglutination reaction inrelation to a patient sample;

FIG. 2 is a pictorial representation of a portion of the prior art testcard of FIG. 1, as contrasted with columns of a test element design thatis made in accordance with a first embodiment; and

FIG. 3 depicts an immunodiagnostic test element made in accordance witha second embodiment following testing wherein agglutination reactionsformed within columns of the test element can be graded by way ofindicia provided on the test element.

DETAILED DESCRIPTION

The following description relates to an improved immunodiagnostic testcard or cassette that includes indicia to enable a user and/or anautomated apparatus to better visualize and grade sample agglutinationreactions for purposes of blood bank applications such as antibodyscreening and identification, ABO blood grouping and Rh phenotyping,reverse serum grouping, direct antiglobulin testing and antigen typing,among other uses. This indicia also provide a means to more effectivelycontrol and maintain aspects relating to the manufacture of animmunodiagnostic test card or cassette. It will be readily apparent tothose of sufficient skill that there are available variations andmodifications that are within the inventive aspects discussed herein.For example and in the examples described herein, the patient sampletested is red blood cells (RBCs) or sera, though the patient sample caninclude other body fluids, such as but not limited to amniotic fluid,spinal fluid, urine, plasma, and serum or any other body fluid that iscapable of producing an agglutination reaction. In addition, severalterms are used throughout in order to provide a suitable frame ofreference with regard to the accompanying drawings. These terms are notintended to be limiting of the concepts covered by the claims of thisapplication, except where so specifically indicated.

Referring to FIG. 1 and for purposes of background, there is shown aprior art immunodiagnostic test element, in this case a gel card 10. Thegel card 10 shown is defined by a substantially flat planar member 14 orsubstrate that further includes a plurality of microtubes or columns 18,each being arranged in parallel and in a vertical orientation relativeto top and bottom surfaces 22, 26 of the test card. The material of thegel card 10 and the supported microtubes 18 themselves is notnecessarily critical. In one embodiment, the microtubes 18 are formedfrom a transparent plastic material such as polyethylene, polystyrene orPVC and are integrally manufactured by means of blister packaging whilethe substrate is manufactured from polystyrene or a similar structuralmaterial. Each of the microtubes 18 are defined by a substantiallycylindrical well 30 that includes an open-ended upper cylindricalportion 34 extending downwardly from the top surface 22 of the card 10to a closed lower cylindrical portion 42. The upper cylindrical portion34 has a somewhat constant diameter that extends to an inwardly taperingor transitional portion 36 intermediate the upper and lower portionswherein the lower portion 42 includes a diameter that is smaller thanthat of the upper portion. The immunodiagnostic gel card 10 depicted inFIG. 1 includes a total of six (6) vertically disposed columns(microtubes), though this number can be suitably varied depending on thetest to be performed.

The gel card 10 further includes a product label 15 that is adhesivelyattached to one facing side of the card, the label including relevantinformation such as the type of card, lot information and expirationdate and having both visual readable and machine readable sections, suchas bar-coded section 16.

Provided within each of the defined test chambers 30 is a quantity oftest material 46 that produces a reaction with the patient sample andprovides a means for separation of agglutinates. In the specific gelcard illustrated in FIG. 1, this column is approximately 15 mm long and4 mm wide. Each column contains a dextran acrylamide gel prepared in abuffer solution, such as LISS or saline. The gels may also contain otherelements: preservatives such as sodium azide, sedimenting agents such asbovine serum albumin, and in some cases, specific reagents such asanti-IgG or other RBC-specific antisera (ABO and D). In the instancewhen reagents are added, they are dispersed throughout the length of thegel column. As such, the gel column is approximately 75 percent packedgel and 25 percent liquid. The testing material 46 is typically providedby the manufacturer wherein a pierceable foil wrap is used to cover thetop side of the card and each of the microtubes 18, sealing thecontents. In an automated apparatus, the foil wrap is pierced andpatient sample and possibly reagents are added and then the test card iscentrifuged to accelerate an agglutination reaction.

As noted, this test element 10 is exemplary of those that include atleast one supported column that includes a test material and is capableof producing an agglutination reaction with regard to a sample. To thatend examples of test cards or cassettes, including features relating tothe inert testing material and related test processing, are described ingreater detail in U.S. Pat. Nos. 5,338,669, 5,460,940, 5,512,432, and6,114,179, the entire contents of which are herein incorporated byreference. The reaction can occur in the top of the test chamber 34 oron the lower portion 42 wherein the strength of reaction is measured bythe position of the formed agglutinates in the gel column or by the lackof agglutinates.

Following testing of a test card, such as described by the foregoingpatents and as depicted in FIG. 1, there are various grades ofagglutination reactions ranging between a strong positive reaction and astrong negative reaction, with gradients therebetween. As notedpreviously, a grading system is used with regard to a resultingagglutination reaction with regard to RBC agglutinates, labeledgenerally by reference numeral 50, that are trapped anywhere in the gelcolumn. Positive reactions can be graded from 0 to 4+. Morespecifically, a 4+ reaction is indicated by a solid band of RBCs on topof the gel. A 3+ reaction displays agglutinated RBCs in the upper halfof the gel column. A 2+ column is characterized by RBC agglutinatesdispersed throughout the length of the column. A 1+ column is indicatedby RBC agglutinates mainly in the lower half of the gel column with someagglutinated RBCs 50 being pelleted at the bottom of the column.Negative (0) reactions are characterized by a pellet of RBCs on thebottom of the microtube with no agglutinates along the length of thecolumn. In the course of testing, the resulting reaction can be highlypositive; that is, all or most of the formed agglutinate is disposedabove the inert material layer, or highly negative; that is, in which noagglutination results and all of the cells is located at the bottom ofthe microtube as a pellet. Determinations of the separate reactions canbe made visually by one or more persons or through machine vision suchas provided on various automated apparatus, such as the ProVue®manufactured by Ortho-Clinical Diagnostics, Inc., using theabove-depicted test card 10, the machine vision system including anillumination system.

Referring to FIG. 2, there is shown an immunodiagnostic test card 70that includes for comparison purposes a first series of supportedcolumns 74 made in accordance with the prior art and a second series ofsupported columns 78 having indicia in the form of indicator marks 82.The marks 82 can be provided for the purpose of defining reaction gradesas described in greater detail in a following section. According to thisspecific embodiment, the supported columns and test substrate are eachmade from a moldable plastic material such as polystyrene, PVC, orpolyethylene. Each of the columns 74 and 78 are similarly constructed asdescribed with regard to FIG. 1 including an upper, open-endedcylindrical portion 34, a transitional intermediate portion 36 and aclosed lower cylindrical portion 42. The indicator marks 82 according tothis embodiment are specifically formed with each of the plastic columns74 and are molded therein, though the marks can otherwise be added, forexample, by laser etching, printing or other similar means. The indiciaaccording to this embodiment are a set of parallel horizontal lines 82having a known separation therebetween and having a predetermined lengthextending across a portion of the width of each column. This indicia canalternatively be provided immediately adjacent each of the columns, forexample, as shown by a similar set of horizontal spaced lines shown as86. It will be readily apparent that other forms of indicia havingvarious shapes and lengths can be used linearly spaced “tick marks”,though other indicia such as those having trapezoidal, circles,shouldered or other form or shape can be utilized. Additionally, atleast some of the indicia can be colored to further permit recognition,either visually or by machine vision.

In addition and according to this embodiment, each of the lines 82, 86are raised above the column surface so as to create distinct relief fromthe smooth card surface that is more easily discerned by theillumination system of an automated machine vision apparatus (not shown)as a visual datum, for example, using the herein described lines ofknown length and separation. This indicia assists the image processingalgorithms of an automated apparatus locate the columns of the card inthe visual field. Moreover, the raised marks also breaks off the smoothsurface of the card and provides a transition point for light emittedfrom the illumination system of the apparatus to reflect from. Thisreflection will be more easily observed as an edge by the imageprocessing software of the automated apparatus. Alternatively, the lines82 and 86 could be recessed or depressed in relation to the surroundingcolumn surface or that of the substrate.

A test card 100 made in accordance with a second embodiment is depictedin FIG. 3. For purposes of this discussion, it should be noted thatsimilar parts are labeled with the same reference numerals. The testcard 100 is defined by a planar substrate 14 that supports a pluralityof microtubes 18. The microtubes 18 like the preceding commonly includean upper cylindrical portion 34, an inwardly tapering transitionalportion 36 and a lower cylindrical portion 42, the latter defining a setof test columns representative of those provided on the immunodiagnostictest card of FIG. 1, but further including indicia in the form ofindicator marks. According to this embodiment, the indicia arerepresented by a series of horizontal evenly spaced parallel lines 102,each of the lines being raised and provided on the exterior surface ofeach of the columns and planar substrate 14. According to thisembodiment, the lines 102 are formed by laser etching though can beadded through other suitable means. Each of the indicator lines 102 areevenly spaced with respect to one another and define separation orgradation zones therebetween for purposes of either describing orgrading an agglutination reaction involving at least one column of thetest card or for purposes of manufacture of the card for purposes ofadding gel material thereto and providing a datum or reference for thatpurpose. In addition, the use of indicator marks, such as thosedescribed herein, can also provide an indication following manufactureand prior to use, as to whether there have been evaporative effects asto the contents of any of the microtubes. It has been determined that inspite of the foil wrap that evaporation can still occur over time andtherefore the indicator marks provide yet another function. The testcard 100 illustrated includes six (6) microtubes 18, thereby defining atotal of 6 test columns.

A working example is herein described for purposes of clarity forpurposes of performing a direct antiglobulin test (DAT) for thedetection of blood cells that are coated with IgG and/or complement dueto an in vivo sensitization using the test card 100 of FIG. 3. The testis performed using an ID-MTS gel card manufactured by Micro TypingSystems, Inc. For purposes of this test card and according to thisexample, five ml of Sephacryl, 200 Gel (Pharmacia) is washed twice insaline solution. The gel particles have a diameter of between about 10and 200 microns. After centrifuging, (5 min, 1250×g) the supernatant isdiscarded and the sediment is filled up to 4.5 ml with isotonic imidazolbuffer (0.014 mol/l imidazol 0.085% NaCl), pH 7.6. PolyspecificAnti-Human Globulin (Anti-IgG, -C3d) is then added to the abovesuspension and is used for detecting IgG and/or complement bound topatient red blood cells. The suspension is mixed well and is ready foruse in this form. Approximately 35 μl of the above solution is thenplaced in each of the test chambers of the gel card, the latter beingmade from polyethylene (the micro-tubes of this example being ET-29 mM,sold by Milana SA, Geneva, Switzerland). The inert particles settle tothe bottom of each microtube 18 within a few minutes.

In terms of the DAT test procedure and using a gel microtube 18 thatcontains Anti-IgG, -C3d as described and 50 μl of suspended RBCs at an0.8 percent concentration is added to the reaction chamber of themicrotube. The mixture is centrifuged for 10 min at approximately 70×g.During centrifugation, RBCs in the reaction chamber are pulled into thegel column. Sensitized RBCs will pass through the upper part of the gelcolumn and agglutinate in the presence of the anti-IgG. AgglutinatedRBCs are too large to pass through the gel matrix and therefore theseagglutinates become trapped at various places along the length of thegel column, depending on the size of the agglutinates. UnagglutinatedRBCs slip easily through the gel column and pellet at the bottom of themicrotube.

The test card 100 depicted includes columns that permit direct Coombstesting, as described above, and control sampling using the six (6)columns. Only the direct AntiGlobulin Test (DAT) has been describedherein. More specifically and according to the test card depicted inFIG. 3, the columns of the test card 100 have undergone testing and theresulting agglutination reactions can be graded, using the indicia. Aspreviously noted, each of the horizontally disposed indicator lines 102are spaced from one another, thereby forming a plurality of gradation orseparation zones that can be utilized either through direct vision by auser or through machine vision, such as described in EP 063774481, theentire contents of which are herein incorporated by reference. Forpurposes of this discussion, the position of the agglutinates and hencethe reaction in each gel column can be easily graded based on theirrelative position. Nominally, the reactions can be scaled as follows:First, the presence of all agglutinates above the uppermost indicatorline provides the indication of a strong positive reaction and a gradeof 4+. On the other hand, the formation of a pellet at the bottom of amicrotube with no formed agglutinates anywhere in the column indicates anegative reaction (0) has occurred. Each of the foregoing are usuallyfairly easy to associate. By virtue of the indicator lines 102, scalingin addition can be established. More specifically, the presence offormed agglutinates in the zone between the uppermost indicator line 105and the center indicator line 107 in spite of the formation ofagglutinates above the gel layer, provides an indication that a weakerpositive reaction has taken place. Likewise, the presence of anyagglutinate in the zone between the lowermost indicator line 109 and thecenter indicator line 107, despite the formation of a pellet at thebottom of the microtube indicates that a very weak positive reaction hasoccurred.

More specifically and by way of the example test card in FIG. 3 andbeginning from the far left side of the card and in microtube 104, it isapparent that a strong positive reaction has taken place wherein theformed agglutinates 128 are entirely formed above the inert material 46as well as above the uppermost indicator line 105. The lack of anyagglutinates below the uppermost indicator line provides an indicationof a strongly positive 4+ reaction grade. In the adjacent second column108, and though a portion of the formed agglutinates 130 are locatedabove the uppermost indicator line 105, another portion is found in thezone formed between the uppermost and the center indicator lines 105,107. As noted above, the latter presence of agglutinates indicates thatreaction that is more weakly positive than that in the foregoing firstcolumn has occurred. As a result, this reaction has a grade of 3+. Inthe third adjacent column 112, formed agglutinates 132 are spread ordispersed across each of the zones defined by the three indicator lines105, 107, 109. This general dispersion indicates the existence of aweaker reaction than that indicated in the second column 108 and definesa 2+ reaction. In the fourth column 116, formed agglutinates 136 aresolely found in the zone defined between the lowest indicator line 109and the center indicator line 107. No agglutinates are found above thecenter indicator line 107. The presence of agglutinates 136 in this zoneindicates a 1+ reaction. Finally and in the fifth and sixth columns 120,124, no formed agglutinates have formed with a pellet of cells 138settling at the bottom of each column below the lowermost indicator line109, thereby indicating that a negative or (0) reaction has occurred ineach instance.

The indicia used are lines, raised or otherwise, as depicted accordingto FIGS. 2 and 3. However and as previously noted, it will be readilyapparent that other forms or types of indicia can be used, including,but not limited to circles, trapezoids, square shouldered lines, and thelike. In addition, this indicia can be clear or alternatively colored inorder to accent between agglutinate and the test matrix and can also beformed either above or below the surrounding surface.

Indicia, such as shown in FIGS. 2 and 3, can also be used in terms ofthe manufacture of immunodiagnostic test cards. By placing indicia inrelation to each transparent microtube, a determination can be made asto the quantity of inert gel, bead or similar material being added tothe confines of each test chamber as well as other manufacturingprocesses, for example, the proper placement of a product label 15 orthe placement of the coded section 16 of the label.

PARTS LIST FOR FIGS. 1-3

-   10 immunodiagnostic test card-   14 flat planar member or substrate-   15 label-   16 machine-coded section-   18 microtubes-   22 top surface, card-   26 bottom surface, card-   30 cylindrical well or chamber-   34 upper cylindrical portion-   36 inwardly tapering or transitional portion-   42 lower cylindrical portion-   46 material, test-   50 agglutinates-   70 immunodiagnostic test card-   74 columns-   78 columns-   82 indicator marks-   86 indicator marks-   100 test card-   102 indicator lines-   104 first column-   105 uppermost indicator line-   107 center indicator line-   108 second column-   109 lowermost indicator line-   112 third column-   116 fourth column-   120 fifth column-   124 sixth column-   128 formed agglutinate-   130 formed agglutinate-   132 formed agglutinate-   136 formed agglutinate-   138 pellet

Though the concepts that have been described relate to specificembodiments and related methods, it will be readily apparent to those ofsufficient skill that there are variations and modifications embodyingthe inventive ambits as recited in the following claims. As noted, thetests and test elements described herein are merely exemplary asliterally any form of blood banking or other serological and sampletesting can be similarly conducted embodying the concepts describedherein using any test element capable of forming an agglutinationreaction. In addition, the lines defined described above are alsoexemplary, wherein the spacing and configuration can suitably be varied,for example, depending on the form of test and design of the testchamber used.

1. An immunodiagnostic test card, said test card comprising: a planarsubstrate; and a plurality of transparent columns supported by saidplanar substrate, each of said transparent columns retaining a quantityof one of beads and gel material for trapping formed agglutinates froman agglutination reaction created by a patient sample and at least onereagent, each of said transparent columns further including indicia,said indicia including a set of at least three reaction grade markingsdisposed in relation to the beads and gel material, wherein each of saidplurality of transparent columns is initially filled with said quantityof one of beads and gel material, said markings each being spaced inrelation to one another to create a plurality of adjacent visiblegrading zones to permit the assignment of a reaction grade based on theposition of the majority of formed agglutinates in each transparentcolumn, said visible grading zones being used collectively in order toassign a reaction grade based on the position of the majority of formedagglutinates with respect to one of said visible grading zones.
 2. Atest card as recited in claim 1, wherein said plurality of transparentcolumns is made from a moldable plastic material, in which said indiciaare molded directly into said at least one transparent column.
 3. A testcard as recited in claim 2, wherein said indicia includes at least oneraised or depressed surface, defining said grade markings.
 4. A testcard as recited in claim 2, wherein said indicia includes a plurality ofraised or depressed surfaces defined by a series of parallel spacedlines having separations therebetween, said lines being disposed on saidat least one transparent column in relation thereto to define saidgrading zones such that the position of formed agglutinates within oneor more of said grading zones defines a grade of reaction.
 5. A testcard as recited in claim 1, wherein said indicia are provided adjacentto at least one transparent column.
 6. A test card as recited in claim1, wherein said indicia are one of at least etched and printed onto saidcard.
 7. A test card as recited in claim 1, including an indicator fordetermining evaporative effects of liquid retained in at least one ofsaid plurality of transparent columns, said indicator including at leastone of said markings.
 8. A test card as recited in claim 1, furtherincluding a label attached to said test card, wherein at least one ofsaid indicia provides a datum for locating said label on said card.
 9. Atest card as recited in claim 1, including a fill indicator forinitially filling each transparent test column with said one of beadsand gel material, and in which at least one of said indicia is used assaid fill indicator.